Sandbox Reserved 350


 * Protein: cHuman Coagulation factor V, 1czv

=Introduction=

Coagulation Factor V originally studied in 1987 by William H. Kane, Akitada Ichinose, Frederick S. Hagen and Earl W. Davie, out of University of Washington, Seattle. Coagulation is a complex cascade of a biological reaction that takes place after an injury causing bleeding, to prevent bleeding; A step of hemostatsis, which facilitates the formation of fibrin. There approximately 30 known factors which play a role in this massive cascade response. A possible explanation for the sheer complexity associated with what is known about this cascade is that tight regulation for the formation of blood clots are crucial.

The role of Human Coagulation Factor V is to act as a cofactor along with Factor X and Prothrombin (Factor II, inactive) to form a Prothrombinase complex activating Prothrobin to Thrombin (FIIa). Thrombin is then able to activate Fibrinogen (Factor I) into Fibrin (FIa), which connects platelets bound at the site of injury; formation of a clot.

It is vital for controlled blood clot formation based on known human diseases/conditions of irregular clotting which are quiet devastating. Two clear aspects of blood clotting involve either under clotting or excessive clotting. The inability to form a clot leads to excessive bleeding from a minor abration known as hemophila. Thrombosis is the second case, where excessive clotting or clotting when no wound is present results in free floating embolisms or thombuses. Floating free in the blood allows the thrombus to lodge itself within the circulatory system, interferring with orgran downstream of the circulating blood. Unusual blood circulation/clotting by a thrombus are causes of heart attacks, strokes and necrosis of tissue.

=Structure & Function= The structure of Human Coagulation Factor V (FV) precursors from a translated polypeptide

to a A1-A2-B-A3-C1-C2 layout which results in the activated (FVa) protein.


 * Heavy A1-A2 Chain


 * Light A3-C1-C2 Chain

The C2 Domain of FVa (FVa-C2) consists of a conserved  β-Barrel framework  acting as a scaffold for  three loops being part of the light chain.



The FVa-C2, which is classified as a   distorted jelly-roll   β-barrel motif, is composed of  eight major antiparallel strands arranged into two   β-sheets of five and  three strands  packed against one another.

Salt bridges located within the "upper" segment '''  (Asp61-Arg134). The C2-Domain of Human coagulation factor is homologous to a larger family of adhesion proteins; Discoidin, but not related to synaptotagmin-like C2 domains.

 The Three Loops


 * Apex 1—Ser21-Trp31; containing Indole moieties able to form hydrogen bonds (Involving two consecutive Trp 26 & 27).


 * Apex 2—Asn39-Asn45; capped with a basic residue able to form hydrogen bonds (Arg43).


 * Apex 3—Gly75-Tyr84; Hydrophobic Loop (Leu79).

The apexes of these  three loops within the C2 domain, are able to create a deep groove lined by hydrophobic  (Trp31, Met83) and polar residues  (Gln48, Ser78), as seen and consisting the <scene name='Sandbox_Reserved_350/Expriment3/3'> Open Form of FVa-C2. This groove is seen as the primary membrane-binding site of the C2-Domain.

A second dimeric crystal form of FVa-C2, packed through the free edges of S6 strands, presenting a different Leu104-Val109 loop, suggests capabilities of adopting a "Closed Form". In contrast to the "Open Form" of FVa-C2; when looking at the loops 1 and 3 are tilted towards the interior of the groove. This change is considered due to a twist around Gly28 cause it to be deformed (pseudo). In general there is a narrowing of the entrance to the shallow inner loop groove, particularly the critical Gln48 carboxamide; Taking place due form the concerted tilting/ "twisting" of the main chain atoms, shifting up to ~7Å and a 12Å displacement of the Trp27 moiety &rarr; shifting closer to the other two loops. Once shifted closer, the groove seen in the Open Form is covered by a hydrophobic ridge of Trp27, Trp27 and Leu79, and now in the Closed Form with a smaller, 370Å hydrophobic surface compared to 520Å.

The three loops are described by Macedo-Ribeiro et al. to protrude like spikes from the bottom of the barrel in monomeric FVa-C2. It is also worth noting that spike (1) & spike (3) are separated by β-hairpin structures and spike (2) is described as a wider irregularly loop comparatively. These three loops extending from the C2 domain, are all linked to each other, and to three shorter loops by an intricate H-bonding network which extends to residues at the bottom of the β-barrel.

The overall Barrel structure is closed at the top and bottom by straight segments, giving it an overall spherical shape with a flattened upper surface.

Functionality of Human Coagulation Factor V (FV), as most proteins is strongly correlated to the conformation of the overall structure. As noted above in the structural section, two structural forms of Human Coagulation Factor V C2 Domain were crystallized. These two structures were distinct from each other based on a conformational change present, alternating between what was described as a Open and Closed form. This change from close to open by exposing this groove results due to the kind of environment Human Coagulation Factor V finds itself in, and ultimately the source of its function. This confers that FV is not an enzymatically active protein, but instead acts a cofactor part of the larger

The role of Human Coagulation Factor V, is act as a cofactor, enhances the ability of factor Xa to generate from prothrombin once activated (Fva). It is known that FV is activated in a positive feedback mechanism by α-thrombin and aided in conjunction with Human Coagulation Factor Xa, and inhibited by Active Protein C (1aut). Originally, the activation of FV to Fva was understood to require the excision of B segment between the heavy and light chain at Arg-1018 and Arg-1545. The peptide as a whole remains united via the disulfide linkage connecting the N and C Terminus and interactions with calcium ions. In 1999, the crystallization of FV in both Fv and Fva was identified providing further insight into the quaternary structure of FV and the underlining mechanism by which the protein functions. This mechanism proposed has three novel points, which were over-looked based on assumed knowledge from previous studies modeling the mechanism for FV after the well characterized mechanism of vitamin K-dependent Coagulation Factors; VII (1dan) , IX (1pfx) , X (1c5m) and Protein C

The Mechanism Proposed differs from the previous work, showing Ca2+-Independent stereospecific binding to phospholipid membranes, based on;

(1) Immersion of Hydrophobic residues at the apices of loops in apolar membrane core.

(2) Specific Interactions with phosphatidylserine head groups in the groove enclosed by these loops.

(3) Favourable electrostatic contacts of basic side chains with negatively charged membrane phosphate groups.

Detailed Proposed Mechanism
For stereospecific and cooperative association with (PʟS)-rich membranes.

(1) FVa, with C2 domain in its closed form, approaches acidic membranes directed by protein-membrane electrostatic interactions.

(2) One or two (PʟS) molecules bind to anchoring points Arg150 or Gln48 in small groove of closed from → triggers widening of the groove and conversion to the open form.

(3) [(Fig. 5a.)]–A (PʟS) molecule occupies the opened specificity pocket.

(4) Now the unfolded hydrophobic spikes, perforate the polar membrane surface; Trp26, Trp27 and Leu79 side are immersed into the apolar core.

(5) Bottom of β-Barrel contacts negatively charged phosphate head groups on the membrane through favourable ionic interactions with basic residues located in the spikes and neighboring loops.


 * The C2 domain of FVa is essential for binding to phosphatidyl-ʟ-serine (PʟS). This is because the spikes and neighboring loops are highly connected and several continuous PʟS molecules would be necessary for association with spike unfolding and membrane insertion for a cooperative mechanism.  This assures FVa-C2 binds only to cell membranes with the concentration of PʟS exceeding a critical threshold level and therefore selectively target FVa to procoagulated, PʟS-rich surfaces.

Experimental Evidence
Binding of FVa to a few acidic, lipid-specific sites results in substantial protein conformational changes.


 * A3 Domain interacts with phosphatidyl-choline.
 * Contributions from hydrophobicity of the heavy chains; A1 and A2.
 * Speculation of further conformational changes as associated with membrane, where the C2 Domain rotates to bring spike 1 and the Trp-rich surface (flat and extended) covering the front-side of the β-Barrel in contact with the phospholipid membrane [(Fig. 5b.)].

=Finer Crystallography Details= X-Ray Diffraction of the C2 Domain of Human Coagulation Factor V (1czv)

Resolution Method:Structure Replacement

Resolution: 2.40Å


 * High: 2.40Å
 * Low: 8.00Å

Condition;


 * pH: 10.00


 * Temperature: 289.0K


 * Number of Crystals used: 1

Geometry: Unit Cell Length        :           Angle


 * a= 86.52Å                   :            α= 90°


 * b= 70.54Å                   :            β= 90°


 * c= 60.58Å                   :            γ= 90°

Radiation Source: Rotating Anode

Wavelength OR Range: 1.5418Å

Detector Type: Image Plate

=References=